Project Summary Acetylcholine (ACh) is an ancient signaling molecule found in many organisms including bacteria, protozoa, plants, and animals. ACh detection through nicotinic and muscarinic receptors occurs in many human organs besides the nervous system but these much less studied than its neuronal function. One of the non-synaptic functions of ACh signaling is in cell migration during normal development. For example, smooth muscle cells, epithelial keratinocytes, and mesenchymal stem cells migrate in response to ACh, but a detailed mechanistic understanding of how this signaling is utilized in migration, particularly in vivo, is not available. We have developed a unique system with which to study the role of Ach signaling in cell migration in vivo. The C. elegans linker cell (LC) is a specialized epithelial cell that leads the migration of the developing male gonad. We found through single-cell transcriptomics that the LC expresses many neuronal receptors and ion channels, leading to the hypothesis that the LC uses neuronal cues for its migration. We will investigate the role of acetylcholine signaling in the LC because we observed migration defects in mutants lacking particular muscarinic and nicotinic receptors. We will initially focus on the only muscarinic receptor expressed in the LC, GAR-3, that we found to activate changes in cell orientation. We will in parallel investigate the function of the multiple nicotinic receptors in LC migration and potential cooperation between the receptor types. The LC reverses its orientation from the posterior to anterior in response to excess ACh signaling induced by treatment with the acetylcholinesterase inhibitor aldicarb, an assay we will use to identify the downstream components of the GAR-3 pathway in the LC. Since downstream targets of the branching gar-3 signaling pathway have been identified in other contexts (e.g., synaptic transmission), we will test mutants of those genes to identify the specific downstream pathway used to modulate LC orientation. To investigate the role of ACh distribution, we will reprogram the fate of neurons in the ventral nerve cord (VNC) to create artificial anterior/posterior ACh gradients to investigate how ACh distribution and amount affects LC migration. We will collaborate to use Correlation Electron Microscopy to examine GAR- 3 localization relative to the ultrastructure of LC and surrounding tissue at high resolution. We will reuse these assays to study potential effects of six nicotinic receptor subunits.